A neutral pH microclimate had been shown at the luminal surface of the large intestine. The aim was to estimate to what extent fluxes of propionic acid/propionate are affected by changes of the luminal pH when this microclimate is present, largely reduced or absent. Fluxes of propionic acid/propionate (J(Pr)) across epithelia from the caecum, the proximal and the distal colon of guinea pigs were measured in Ussing chambers with and without a filter at the luminal surface. With bicarbonate and with a neutral or an acid pH of mucosal solutions (pH 7.4 or 6.4), mucosal-to-serosal fluxes (J(ms)(Pr) ) were 1.5 to 1.9-fold higher at the lower pH, in bicarbonate-free solutions and carbonic anhydrase (CA) inhibition 2.1 to 2.6-fold. With a filter at the mucosal surface and with bicarbonate containing solutions, J (ms) (Pr) was not or only little elevated at the lower pH. Without bicarbonate J(ms)(Pr) was clearly higher. We conclude that the higher J(ms)(Pr) after luminal acidification is due to vigorous mixing in Ussing chambers resulting in a markedly reduced unstirred layer. Therefore, an effective pH microclimate at the epithelial surface is missing. J(ms)(Pr) is not or is little affected by lowering of pH because in the presence of bicarbonate the filter maintains the pH microclimate. However, in bicarbonate-free solutions J(ms)(Pr) was higher at pH 6.4 because a pH microclimate does not develop. Findings confirm that 30-60% of J(ms)(Pr) results from non-ionic diffusion.